TAILIEUCHUNG - Báo cáo khoa học: Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor (R2F) and a stable tyrosyl radical (Y•) involved in ribonucleotide reduction

Ribonucleotide reduction, the unique step in the pathway to DNA synthe-sis, is catalyzed by enzymes via radical-dependent redox chemistry involv-ing an array of diverse metallocofactors. The nucleotide reduction gene (nrdF) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenesribonucleotide reductase was reintroduced into strain C. | ỊFEBS Journal Homologous expression of the nrdF gene of Corynebacterium ammoniagenes strain ATCC 6872 generates a manganese-metallocofactor R2F and a stable tyrosyl radical Ya involved in ribonucleotide reduction Patrick Stolle1 Olaf Barckhausen1 Wulf Oehlmann1 Nadine Knobbe2 Carla Vogt2 Antonio J. Pierik3 Nicholas Cox4 Peter P. Schmidt4 f Edward J. Reijerse4 Wolfgang Lubitz4 and Georg Auling1 1 Institut fur Mikrobiologie Leibniz Universitat Hannover Germany 2 Institut fur Analytische Chemie Leibniz Universitat Hannover Germany 3 Institut fur Zytobiologie Philipps Universitat Marburg Germany 4 Max-Planck-Institut fur Bioanorganische Chemie Mulheim Germany Keywords Corynebacterium ammoniagenes EPR homologous expression manganese-tyrosyl metallocofactor ribonucleotide reductase Correspondence G. Auling Institut fur Mikrobiologie Leibniz Universitat Hannover Schneiderberg 50 D-30167 Hannover Germany Fax 49 511 762 5287 Tel 49 511 76 5241 E-mail auling@ Present address Olaf Scheibner Thermo Fisher Scientific GmbH Bremen Germany fDeceased 2008 Received 21 February 2010 revised 7 September 2010 accepted 17 September 2010 Ribonucleotide reduction the unique step in the pathway to DNA synthesis is catalyzed by enzymes via radical-dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene nrdF encoding the metallocofactor containing small subunit R2F of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac-promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron 30 1 high activity 69 pmol 2 -deoxy-ribonucleotide-mg_1-min-1 and distinct absorption at 408 nm characteristic of a tyrosyl radical Y which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of

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